Bacterial Phosphoenolpyruvate-Dependent Phosphotransferase System: Mannitol-Specific EII Contains Two Phosphoryl Binding Sites per Monomer and One High-Affinity Mannitol Binding Site per Dimer

The amino acid composition and sequence of EIIMtl is known. This information was combined, in the present study, with quantitative amino acid analysis to determine the molar concentration of the enzyme. The stoichiometry of phosphoryl group incorporation was then determined by phosphorylation of enzyme II from [14C]phosphoenolpyruvate (pyruvate burst procedure). The native, reduced enzyme incorporated two phosphoryl groups per monomer. Both phosphoryl groups were shown to be transferred to mannitol. Oxidation or N-ethylmaleimide (NEM) labeling of Cys-384 resulted in incorporation of only one phosphoryl group per monomer, which was unable to be transferred to mannitol. The number of mannitol binding sites on enzyme II was determined by centrifugation using Amicon Centricon microconcentrators. The reduced unphosphorylated enzyme contained one high-affinity binding site (KD = 0.1 µM) per dimer and a second site with a KD in the micromolar range. Oxidation or NEM labeling did not change the number of binding sites.